RESUMEN
AIM: To investigate the possible mechanism by which hepatitis B virus X protein (HBx) mediates apoptosis of HepG2 cells. METHODS: HBx expression vector pcDNA3.1-X was transfected into HepG2 cells to establish an HBx high-expression cellular model as pcDNA3.1-X transfected group. The pcDNA3.1-X and pSilencer3.1-shHBX (HBx antagonist) were cotransfected into HepG2 cells to establish an HBx low-expression model as RNAi group. Untransfected HepG2 cells and HepG2 cells transfected with negative control plasmid were used as controls. Apoptosis rate, the expression of Fas/FasL signaling pathway-related proteins and the phosphorylation levels of MLK3, MKK7 and JNKs, which are upstream molecules of death receptor pathways and belong to the family of mitogen-activated protein kinases (MAPKs), were measured in each group. RESULTS: Compared with HepG2 cell group and RNAi group, apoptosis rate, the expression of Fas and FasL proteins, and the activation of MLK3, MKK7 and JNKs were increased in the pcDNA3.1-X transfected group. The activation of JNKs and expression of FasL protein were inhibited in the pcDNA3.1-X transfected group when treated with a known JNK inhibitor, SP600125. When authors treated pcDNA3.1-X transfected group with K252a, a known MLK3 inhibitor, the activation of MLK3, MKK7 and JNKs as well as expression of FasL protein was inhibited. Furthermore, cell apoptosis rate was also significantly declined in the presence of K252a in the pcDNA3.1-X transfected group. CONCLUSION: HBx can induce HepG2 cell apoptosis via a novel active MLK3-MKK7-JNKs signaling module to upregulate FasL protein expression.
Asunto(s)
Apoptosis/fisiología , Proteína Ligando Fas/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , MAP Quinasa Quinasa 7/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Transactivadores/metabolismo , Animales , Caspasas/metabolismo , Activación Enzimática , Inhibidores Enzimáticos/metabolismo , Células Hep G2/fisiología , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/genética , MAP Quinasa Quinasa 7/genética , Quinasas Quinasa Quinasa PAM/genética , Transactivadores/genética , Proteínas Reguladoras y Accesorias Virales , Receptor fas/metabolismo , Proteina Quinasa Quinasa Quinasa 11 Activada por MitógenoRESUMEN
OBJECTIVE: To investigate the effect of hepatitis B virus X gene on the apoptosis in X gene-transfected HepG2 cells. METHODS: HBX gene eukaryon expression vector pcDNA3.1-X was transfected into HepG2 cells by lipid-mediated transfection to establish HepG2/HBX cell model for HBX expression. HepG2 transfected with pcDNA3.1 was used as controls. At 24 h, 48 h, 72 h and 96 h after transfection, cell apoptotic rates were detected by flow cytometry. RT-PCR and Western blot were applied to evaluate the expression levels of HBX, Fas, FasL, and the levels of p-JNK and p-c-Jun protein. RESULTS: HBX mRNA was detected in HepG2/ HBX cells 24 h after transfection, and the expression of HBX mRNA level was gradually up-regulated after transfection (P < 0.05); whereas no expression of HBX gene in the control cells. At 24 h, 48 h, 72 h and 96 h after transfection, all of the apoptotic rate in HepG2/HBX group were much higher than those in the controls (P < 0.05). The expression levels of Fas and FasL protein as well as the levels of p-JNK and p-c-Jun protein were significantly higher than those in the controls (P < 0.05). A positive correlationship was observed between the expression of HBX mRNA level and the hepatocyte apoptotic rate (P < 0.05), the same correlation of HBX mRNA level with the levels of Fas, FasL, p-JNK and p-c-Jun were also observed. (P < 0.05). CONCLUSION: HBX can induce hepatocyte apoptosis by up-regulating the levels of p-JNK and p-c-Jun to promote the expression of FasL.
